The Effects Of QSOX1 And Its N-glycosylation On Recurrence And Metastasis Of Hepatocellular Carcinoma

In 2008, the incidence of liver cancer is more than 748,300 per year, with more than 695,900 deaths. The incidence and deaths of liver cancer in China has been more than half of the world. Hepatocellular carcinoma (HCC) is the most important pathological type of liver cancer. The natural survival time of patients with advanced HCC is only about 3 months. Even after curative resection, patients must face the high rate of metastasis and recurrence. Up to now, treatments for advanced HCC are rare. Target drugs are expensive, and not all patients can benefit. Therefore, effective predictors of metastasis and recurrence can help for not only further dividing HCC patient subtypes, but also early intervention for patients with potential recurrence. Indicators from blood sample could be suit for predicting the prognosis because of its minor invasion. It gives us inspiration that many blood tumor markers are glycoproteins, such as alpha-fetoprotein (AFP) for liver cancer. Screening for prognostic indicators in glycoproteins from plasma is feasible. Dr. Wang Ji applied Lectin chromatography to obtain preoperative N-glycoproteins from serum glycoprotein of patient. Results suggest that expression level of LCH enriched QSOX1 (α1-6 fucosylated QSOX1) in serum of the recurrence group was significantly lower than those without recurrence. These provide a strong support for us to study distribution and biological effects of QSOX1 in HCC. QSOX1 protein has been reported in many types of tumors, but its divergent role in tumor generation and progression has not been elucidated well. Various distribution and extensive functions of QSOX1 may contribute for that. Our current work established strategy on the basis of the previous data of αl-6 fucosylated QSOX1. So we established research methods and intervention models for QSOX1 short form (QSOX1-S), in HCC cell lines and tissues from HCC patients to study the mechanism of the N-glycoprotein in tumor progression. We established mutant protein (no N-glycosylation) overexpression HCC cell line to explore the influence of N-glycosylation on the function of QSOX1-S protein. That may provide valuable information for the production of recombinate protein for the cure of HCC.PART Ⅰ Expression of QSOX1 Protein in HCC cells and Tissues and Its Correlations with Recurrence and MetastasisObjective:QSOX1 protein which has two forms is found to play an important role in multiple tumors. But reports vary in different function of the protein to tumors. We will elucidate the expression of QSOX1 protein and its relation with prognosis in HCC.Methods:Western blot was used to test the protein expression of QSOX1 in different human HCC cell lines, hepatic cell lines and stellate cell line. Immunohistochemical staining of tissue microarray was to elucidate the location and expression levels of QSOX1 in HCC tissues. LCH lectin was used to study the distribution of f-QSOX1 in HCC tissues. Kaplan-Meier curve and Log-rank method was to analyze the correlation between the QSOX1 expression and patient prognosis. Cox regression model was applied to test the independence of the predictor.Results:The two forms of QSOX1 protein distributed in different bands by Western blot. The expression of QSOX1-S, the secreted form (67Kd) of QSOX1, reduced with the invasive ability of HCC cell lines increased. And stellate cell line expressed only low level of QSOX1-S. Immunohistochemical of the tissue microarrays showed the expression level of QSOX1 protein in tumor tissues was significantly higher than the peritumor tissues. Combining the clinical data, the Kanplan-meier curves were analyzed by Log-rank method. It showed that patients with high QSOX1 expression in their tumor tissues had better outcomes. Comparably, patients with high QSOX1 expression in their peritumor tissues had worse prognosis. The diffuse positive expression of QSOX1 in tumor tissue denotes the positive expression of QSOX1-S. The expression level of QSOX1-S in tumor tissues had higher correlation with the recurrence. Cox regression model results suggested that tumor QSOX1-S level is a independent predictor for both survival and recurrence in HCC patients after radical surgery. The proportion of f-QSOX1 in QSOX1-S is higher in tumor tissues than peritumor tissues. The correlation of tumor and peritumor QSOX1-S can predict the prognosis of HCC patients powerfully.Conclusions:Protein expression levels of QSOX1-S in HCC cells were negatively correlated with the invasion ability of the cells. Patients with high QSOX1 expression in their tumor tissues had better outcomes. While, high QSOXl expression in their peritumor tissues had worse prognosis. The expression level of QSOX1-S in tumor tissues had higher correlation with the recurrence. The disparity between expression levels of QSOX1 in tumor and peritumor tissues is caused by the different proportion of f-QSOX1 in QSOX1-S. This combines the expression of QSOX1 in sera and tissues. The combination of tumor and peritumor QSOX1-S levels is a more powerful predictor for prognosis of HCC patients.PART Ⅱ Effect of QSOX1-S Protein on Biological Behavior of HCC CellsObjective:In this study, QSOX1-S knockout and overexpression HCC cell lines established with lentiviral vectors were used to explore the biological effects of the protein on HCC cells in vitro and in vivo. The tumor-related signal transduction pathway influenced by QSOX1-S was studied as well.Methods:QSOX1-S knockout cell line by lentiviral vectors was constructed in Hep3B naturally expressing high level of the protein. QSOX1-S overexpression cell line was constructed in MHCC97H naturally expressing low level of the protein. Western blot was used to verify the expression levels of the two stable transfection cell lines. Meanwhile confocal immunofluorescence was applied to explore the distribution of protein in cells. The effect of QSOX1-S on cell proliferation was tested by CCK8 kit. Cell apoptosis and cell cycle was tested by flow cytometry. Invasion assay of the cells was performed in transwell chamber. The model of liver orthotopic transplantation tumor was established in nude mice to detect the function of QSOX1-S on HCC cells in vivo. Tumor-related signal transduction pathway was tested by Western blot.Results:Results from Western blot showed that QSOX1-S knockout stably transfected cell line was successfully established in Hep3B, QSOX1-S overexpression stably transfected cell line was established successfully in MHCC97-H. Confocal immunofluorescence data revealed that QSOX1-S protein located only in the Golgi apparatus and ER of the control cells but located in any part of the QSOX1-S overexpression cell including cytoplasm and cell membrane, even secretory form. There was no significant change in proliferation of the cell overexpressing QSOX1-S. And proliferation rate of Hep3B-shQSOX1 was significantly higher than the control group. QSOX1-S overexpressing cells had lower anti-apoptosis capacity and had different cell cycle distribution. QSOX1-S overexpression can inhibit the invasion ability of HCC cells, and QSOX1 knockout can promote invasion. In vivo assay showed that QSOX1 knockout significantly increased the volume of the tumor the incidence of lung metastasis; whereas overexpression of QSOX1-S cannot change the tumor volume, but it greatly reduced lung metastasis. Tumor-related signaling pathways EGFR (RTK/Ras/MAPK) and Integrinβ1/FAK/ERK can be suppressed by QSOX1-S.Conclusions:QSOX1-S could inhibit malignant phenotype of HCC cells. QSOX1-S suppresses EGFR and Integrinβ1 signaling pathway.PARTⅢ The Impact of N-glycosylation on the Function of QSOX1-SObjective:Overexpress N-glycosylation-depletion protein QSOX1-SM in HCC cells to study the impact of N-glycosylation on biological functions of QSOX1-S.Methods:Method of point mutation was used to knock 130 N-glycosylation. That can make N-glycosylation lost in the protein. Ligate the above mutant sequence into lentiviral vector to construct stable overexpression cell lines in 97H cells witch overexpresses mutant protein QSOX1-SM or QSOX1-SM2. CCK8 was for detection of cell proliferation, transwell invasion assay for cell invasion, flow cytometry for apoptosis and cell cycle; subcutaneous transplantation tumor model in nude mice was used to study the changes in the biological behavior of HCC cells in vivo. Tumor-related signal transduction pathway was tested by Western blot.Results:QSOX1-SM overexpression cell line was verified. Compared to the control group, cell proliferation, apoptosis, and cell cycle of 97H-QSOX1-SM had no significant difference. Data from nude mice revealed that the volume of subcutaneous tumors and lung metastasis were not significantly different in 97H-QSOX1-SM compared with the control group. The changes of tumor-related signal transduction pathway in 97H-QSOX1-S is different from the 97H-QSOX1-S. In another cell lines, 97H-QSOX1-SM2, the abilities of proliferation, resistance to apoptosis, invasion were all stronger than the control cell line. EGFR, Integrinβ1 and Wnt singaling were upregulated.Conclusions:The alteration in the proportion of f-QSOX1 in QSOX1-S brings significant changes in biological behaviors of tumor cells. That indicates the N-glycosylation is essential for the biological function of QSOX1-S. Recombinant proteins for intervention should be produced in eukaryotic expression systems.Conclusions1. The disparity between expression levels of QSOX1 in tumor and peritumor tissues is caused by the different proportion of f-QSOX1 in QSOX1-S. This combines the expression of QSOX1 in sera and tissues. The combination of tumor and peritumor QSOX1-S levels is a more powerful predictor for prognosis of HCC patients.2. QSOX1-S could inhibit malignant phenotype of HCC cells. QSOX1-S suppresses EGFR and Integrinβ1 signaling pathway.3. Overexpression of the N-glycosylation-depletion protein QSOX1-SM cannot bring the same effect as QSOX1-S. That indicates the N-glycosylation of QSOX1-S may be essential for the formulation of the functional domain to suppress tumor invasion.4. Recombinant proteins for intervention should be produced in eukaryotic expression systems.Novelties1. We firstly reported distribution and function of QSOX1 protein in HCC.2. We identified QSOX1-S protein as an inhibitor of metastasis and recurrence in HCC cells and HCC patients.3. The alteration in the proportion of f-QSOX1 in QSOX1-S brings significant changes in biological behaviors of tumor cells. That indicates the N-glycosylation is essential for the biological function of QSOX1-S.4. Recombinant proteins for intervention should be produced in eukaryotic expression systems.Potential application of this study1. QSOX1-S protein can be used for pathological diagnosis to predict prognosis of HCC patients.2. QSOX1-S protein may be used as an alternative or combinative drug of targeted drug.